1. Field of the Invention
The present invention is in the field of determining a base sequence of a nucleotide strand.
2. Description of the Background Art
The field of DNA sequencing is very active because of the decision to sequence the human genome. Presently available technology for determining a base sequence of a nucleotide strand uses different fluorescence labels on the four nucleotides, adenine, thymine, guanine, and cytosine, during sequencing. The nucleotide is identified by the emission spectrum which is distinct for each of the four probes used for each nucleotide.
The following references describe known DNA sequencing techniques which utilize measurement of fluorescence intensity:
T. Hunkapiller, R. J. Kaiser, B. F. Koop, and L. Hood, xe2x80x9cLarge-Scale and Automated DNA Sequence Determination,xe2x80x9d Science 254:59-67 (1991).
D. B. Shealy, M. Lipowska, J. Lipowski, N. Narayanan, S. Sutter, L. Strekowski, and G. Patonay, xe2x80x9cSynthesis, Chromatographic Separation, and Characterization of Near-Infrared-Labeled DNA Oligomers for Use in DNA Sequencing,xe2x80x9d Analytical Chemistry 67:247-251 (1995).
J. Ju, C. Ruan, C. W. Fuller, A. N. Glazer, and R. A. Mathies, xe2x80x9cFluorescence energy transfer dye-labeled primers for DNA sequencing and analysis,xe2x80x9d Biophysics 92:4347-51 (1995).
J. Ju, A. N. Glazer, and R. A. Mathies, xe2x80x9cEnergy transfer primers: A new fluorescence labeling paradigm for DNA sequencing and analysis,xe2x80x9d Nature Medicine 2:246-49 (1996).
L. M. Smith, J. Z. Sanders, R. J. Kaiser, P. Hughes, C. Dodd, C. R. Connell, C. Heiner, S. B. H. Kent, and L. E. Hood, xe2x80x9cFluorescence detection in automated DNA sequence analysis,xe2x80x9d Nature 321:674-79 (1986).
D. C. Williams and S. A. Soper, xe2x80x9cUltrasensitive Near-IR Fluorescence Detection for Capillary Gel Electrophoresis and DNA Sequencing Applications,xe2x80x9d Analytical Chemistry, 67:3427-32.
S. Wiemann, J. Stegemann, D. Grothues, A. Bosch, X. Estivill, C. Schwager, J. Zimmermann, H. Voss, and W. Ansorge, xe2x80x9cSimultaneous On-Line DNA Sequencing on Both Strands with Two Fluorescent Dyes,xe2x80x9d Analytical Biochemistry 224:117-21 (1995).
K. C. Huang, M. A. Quesada, and R. A. Mathies, xe2x80x9cDNA Sequencing Using Capillary Array Electrophoresis,xe2x80x9d Anal. Chem. 64:2149-54 (1992).
J. M. Prober, G. L. Trainor, R. J. Dam, F. W. Hobbs, C. W. Robertson, R. J. Zagursky, A. J. Cocuzza, M. A. Jensen, and K. Baumeister, xe2x80x9cA System for Rapid DNA Sequencing with Fluorescent Chain-Terminating Dideoxynucleotides,xe2x80x9d Science 238:336-41 (1987).
S. Takahashi, K. Murakami, T. Anazawa, and H. Kambara, xe2x80x9cMultiple Sheath-Flow Gel Capillary-Array Electrophoresis for Multicolor Fluorescent DNA Detection,xe2x80x9d Anal. Chem. 66:1021-26 (1994).
The following references describe known DNA sequencing techniques which utilize measurement of fluorescence lifetime:
M. Sauer, K-T. Han, V. Ebert, R. Muller, A. Schulz, S. Seeger, J. Wolfrum, J. Arden-Jacob, G. Deltau, N. J. Marx, and K. H. Drexhage, xe2x80x9cDesign of Multiplex Dyes for the Detection of Different Biomolecules,xe2x80x9d 1994 SPIE Proc. 2137:762-774.
K-T. Han, M. Sauer, A. Schulz, S. Seeger, and J. Wolfrum, xe2x80x9cTime-Resolved Fluorescence Studies of Labelled Nucleosides,xe2x80x9d Ber. Busenges. Phys. Chem. 97:1728-30 (1993).
K. Chang and R. K. Force, xe2x80x9cTime-Resolved Laser-Induced Fluorescence Study on Dyes Used in DNA Sequencing,xe2x80x9d Applied Spectroscopy 47:24-29 (1993).
J. R. Lakowicz, H. Szmacinski, K. Nowaczyk, K. W. Berndt, and M. Johnson, xe2x80x9cFluorescence Lifetime Imaging,xe2x80x9d Analytical Biochemistry 202, 316-330 (1992).
The dyes described in the literature are based on near infrared probes, energy transfer probes to make the intensities equivalent, and other common fluorophores with visible excitation and emission wavelengths. None of these references mentions the use of metal-ligand complexes in determining a base sequence of a nucleotide strand.
The disadvantages of the currently available technology includes nanosecond decay times, which do not allow suppression of prompt auto-fluorescence, limited photostability, small Stoke""s shifts and spectral overlap between the absorption and emission spectra.
In addition, with nanosecond decay times it is not possible to reject the auto-fluorescence from the samples, which is especially problematic with the low concentrations involved in the DNA sequencing. Furthermore, the use of nanosecond decay time fluorophores for sequencing based on the decay times, as has been proposed by other laboratories, requires complex instrumentation and is thus not likely to be widely utilized.
There is extensive literature regarding the spectral properties of metal-ligand complexes. The following is a list of papers regarding metal-ligand complexes:
Maestri, M., Sandrini, D., Balzani, V., Maeder, U. and von Zelewsky, xe2x80x9cAbsorption Spectra, Electrochemical Behavior, Luminescence Spectra, and Excited-State Lifetimes of Mixed-ligand Ortho-Metalated Rhodium(III) Complexes,xe2x80x9d Inorg. Chem., 26:1323-1327(1987).
Sutin, N. and Creutz, C., xe2x80x9cProperties and Reactivities of the Luminescent Excited States of Polypyridine Complexes of Ruthenium(II) and Osmium(II),xe2x80x9d Inorg. and Organometall. Photochem., Chap. 1, pp. 1-27 (1978).
Hager, G. D., Watts, R. J. and Crosby, G. A., xe2x80x9cCharge-transfer Excited States of Ruthenium(II) Complexes. Relationship of Level Parameters to Molecular Structure,xe2x80x9d J. Am. Chem. Soc., 97;7037-7042 (1975).
Orellana, G. and Braun, A. M., xe2x80x9cQuantum Yields of 3MLCT Excited State Formation and Triplet-Triplet Absorption Spectra of Ruthenium(II) Tris-Chelate Complexes Containing Five- and Six-Membered Heterocyclic Moieties,xe2x80x9d J. Photochem. Photobiol. A. Chem., 48:277-289 (1989).
Harrigan, R. W. and Crosby, G. A., xe2x80x9cSymmetry Assignments of the Lowest CT Excited States of Ruthenium(II) Complexes Via a Proposed Electronic Coupling Model,xe2x80x9d J. Chem. Phys., 59(7):3468-3476 (1973).
Yersin, H. and Braun, D., xe2x80x9cIsotope-Induced Shifts of Electronic Transitions: Application to [Ru(bpy-h8)3]2+ and [Ru(bpy-d8)3]2+ in [Zn(bpy-h8)3] (ClO4)2,xe2x80x9d Chem. Phys. Letts., 179(1,2):85-94 (1991).
Coe, B. J., Thompson, D. W., Culbertson, C. T., Schoonover, J. R. and Meyer, T. J., xe2x80x9cSynthesis and Photophysical Properties of Mono(2,2xe2x80x2,2xe2x80x2-Terpyridine) Complexes of Ruthenium(II),xe2x80x9d Inorg. Chem., 34:3385-3395 (1995).
Lees, A. J., xe2x80x9cLuminescence Properties of Organometallic Complexes,xe2x80x9d Chem. Rev., 87:711-743 (1987).
DeArmond, M. K. and Carlin, C. M., xe2x80x9cMultiple State Emission and Related Phenomena in Transition Metal Complexes,xe2x80x9d Coordination Chem. Rev., 36:325-355 (1981).
Kondo, T., Yanagisawa, M. and Fujihira, M., xe2x80x9cSingle Exponential Decay for the Luminescence Intensity of Ru(bpy)32+ Complex in Langmuir-Blodgett Films,xe2x80x9d Chem. Letts., 1639-1993 (1993).
None of the above references suggest use of metal-ligand complexes in determining a base sequence of a nucleotide strand. Also, the use of metal-ligand complexes is not mentioned in the previous citations on fluorescence and DNA sequencing.
There remains a need in the art for improved methods of determining a base sequence of a nucleotide strand.
In accordance with the present invention, a method for determining a base sequence of a nucleotide strand in a sample includes the step of providing a first fragment of the strand. The emission from metal-ligand complexes may be from mixed singlet and triplet states. We will refer to the emission as fluorescence, though a more precise term may be luminescence. A fluorescent metal-ligand complex is coupled to a first oligonucleotide having a sequence complementary to the first fragment to form a first probe. The first probe is added to a sample that contains the first fragment to form a first mixture containing a first reaction product of the first probe and the first fragment. The first mixture is exposed to an exciting amount of radiation, and the fluorescence of the metal-ligand complex is detected. The first base sequence of the first fragment is identified based on fluorescence of the metal-ligand complex. A second fragment of the strand differing from the first fragment by at least one base is provided. A fluorescent metal-ligand complex is coupled to a second oligonucleotide having a sequence complementary to the second fragment to form a second probe. The second probe is added to a sample that contains the second fragment to form a second mixture containing a second reaction product of the second probe and the second fragment. The second mixture is exposed to an exciting amount of radiation, and the fluorescence of the metal-ligand complex is detected. A second base of the second fragment is identified based on the fluorescence of the metal-ligand complex. The second base sequence is compared to the first base sequence to identify a difference between the first and second sequences to determine a base sequence of the nucleotide strand.
Also in accordance with the present invention the combination of a first probe comprising a fluorescent metal-ligand complex coupled to a first oligonucleotide having a sequence complementary to a first fragment of a nucleotide strand and a second probe comprising a fluorescent metal-ligand complex coupled to a second oligonucleotide having a sequence complementary to a second fragment of the nucleotide strand differing from the first fragment by at least one base, for use in nucleotide sequencing.